水稻剑叶角度主效QTL qFLAG5的克隆与功能分析

The flag leaf angle is regarded as one of the major determinants of rice architecture. To explore gene(s) for flag leaf of rice, primary and secondary F2 populations derived from indica line Changxian R009 and japonica cultivar Sasanishiki were applied for flag leaf angle quantities trait locus (QTL) analysis, and one new major QTL, qFLAG5, was stably detected in the 5.3 cM region between RM18804 and RM18898 on chromosome 5. For high-resolution mapping of qFLAG5, one near-isogenic line, NIL-qFLAG5 (harboring the Changxian R009 qFLAG5 allel in Sasanishiki background) was developed. A large segregation population will be developed by crossing NIL-qFLAG5 with Sasanishiki, by means of linkage analysis using the genotype data of the qFLAG5 and molecular markers, the qFLAG5 can be mapped to a small chromosomal region. Within this region, the candidate gene can be predicted by molecular biological analysis, and the corresponding fragments of Sasanishiki and NIL-qFLAG5 will be sequenced. According to the sequence differences, the candidate gene can be determined. Next we will carry out a complementation test by transforming the Sasanishiki fragment covering the candidate gene into NIL-qFLAG5 to further validate the target gene. Meanwhile, to study the role of qFLAG5 in flag leaf angle development, some molecular biology techniques, such as expression analysis, RNA interference and subcellular localization, will be used for its functional analysis, which can provide theoretical and practical reference for qFLAG5 application in rice plant architecture breeding programs.

水稻剑叶角度是构成株型的重要因素之一。为挖掘水稻剑叶角度基因，本项目利用籼稻昌籼R009，粳稻Sasanishiki的F2、次级F2群体，将新的主效剑叶角度QTL qFLAG5定位在第5染色体RM18804-RM18898之间5.3 cM区段，并构建了Sasanishiki为受体，包含昌籼R009 qFLAG5位点的近等基因系NIL-qFLAG5。为克隆qFLAG5，拟以NIL-qFLAG5与Sasanishiki杂交构建分离群体，利用紧密连锁的标记鉴定重组单株，将qFLAG5精细定位在狭小的染色体区间。通过生物信息学分析确定候选基因并进行测序，根据序列差异确定目的基因。同时，将Sasanishiki的qFLAG5等位基因通过遗传转化导入NIL-qFLAG5中验证目的基因。通过表达分析、RNA干扰和亚细胞定位来验证目的基因的功能，了解其在剑叶角度形成中的作用，为qFLAG5的利用奠定基础。

为克隆水稻叶角QTL qFLAG5，项目组将近等基因系SIL417与背景亲本Sasanishiki杂交构建了次级F2分离群体，共6000株，通过图位克隆的方法将qFLAG5精细定位在206 kb区间；通过候选基因分析和测序，初步确定Os05G0440300为候选基因。通过基因敲除实验，确定Os05G0440300为qFLAG5目标基因。对近等基因系SIL417和背景亲本Sasanishiki目标基因Os05G0440300测序发现，SL417在编码区第2个外显子编码倒数第三个氨基酸的密码子上插入了TA碱基，导致蛋白质翻译提前终止。通过查询水稻基因组注释计划数据库Race Genome Annotaion Project发现，Os05g0440300编码组蛋白去乙酰化酶。激素分析显示背景亲本Sasanishiki响应BR，增大叶角，因此推断Os05g0440300基因可能调控BR合成的基因。对近等基因系SL17和Sasanishiki叶角节点部位进行显微观察，显示大叶角SL17与小叶角Sasanishiki维管束数目发育存在差异，表明SL17叶角节点部位维管束的缺少可能导致支撑能力不足、叶角增大。